PXD051564

PXD051564 is an original dataset announced via ProteomeXchange.

Title	Development of Automated Proteomic Workflows Utilizing Silicon-Based Coupling Agents
Description	This work highlights an automated method for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) and 11-triethoxysilylundecanal (TESU) derived silicone-based coupling agents. From the various coupling strategies we scrutinized, TESB and 4-triethoxysilylbutanoic acid (TESBA), the carboxylic acid derivative, were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness and rapid digestion, while also implementing a more streamlined IEP washing procedure. The physical size of IEPs did not correlate to digestion efficiency, providing insights for potential cost-saving’s in enzyme utilization. Immobilization efficiency was determined to be 50.8 ± 7.7% as validated by Bradford's assay. Furthermore, we adapted the IEP procedure into an automated format with a Bravo liquid handler. This allowed for multiple enzymes, and/or coupling agents to be fabricated at once on a 96-well microplate, in a fraction of the time. The automated trypsin TESB and TESBA IEPs rivaled the classical in-gel digestion method for the analysis of Jurkat cell lysate, achieving similar protein group identifications. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over phenylalanine, tyrosine, tryptophan, and methionine residues. Further, when miscleavages occurred, they tended to be at tyrosine residues, or when two aromatic residues were adjacent. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could serve two roles by functionalizing the silica capillary's inner wall, and simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as little as 15 minutes. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome.
HostingRepository	PRIDE
AnnounceDate	2024-06-10
AnnouncementXML	Submission_2024-06-10_10:48:15.899.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Maor Arad
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	timsTOF SCP

Revision	Datetime	Status	ChangeLog Entry
0	2024-04-18 09:36:04	ID requested
⏵ 1	2024-06-10 10:48:16	announced

10.1016/J.JPROT.2024.105215;

submitter keyword: Capillary Electrophoresis,Immobilized Enzyme Particle, Peptide Mapping, Immobilized Enzyme Microreactor, Automated Liquid Handler, Liquid-Chromatography Mass Spectrometry, Bottom-Up Proteomics

Leonard Foster
contact affiliation	Professor Biochemistry and Molecular Biology Director, Life Sciences Institute
contact email	foster@msl.ubc.ca
lab head
Maor Arad
contact affiliation	University of British Columbia
contact email	maorarad1@gmail.com
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/06/PXD051564

PRIDE project URI

• PRIDE
  1. PXD051564
     1. Label: PRIDE project
     2. Name: Development of Automated Proteomic Workflows Utilizing Silicon-Based Coupling Agents